Molecular Characterization and Expression Profiles of Myrosinase Gene (RsMyr2) in Radish (Raphanus sativus L.)

doi:10.1016/S2095-3119(13)60644-9

Journal of Integrative Agriculture ›› 2014, Vol. 13 ›› Issue (9): 1877-1888.DOI: 10.1016/S2095-3119(13)60644-9

Molecular Characterization and Expression Profiles of Myrosinase Gene (RsMyr2) in Radish (Raphanus sativus L.)

PAN Yan1, XU Yuan-yuan1, ZHU Xian-wen2, LIU Zhe1, GONG Yi-qin1, XU Liang1, GONG Mao-yong1, and LIU Li-wang1

1、National Key Laboratory of Crop Genetics and Germplasm Enhancement/Engineering Research Center of Horticultural Crop Germplasm
Enhancement and Utilization, Ministry of Education/Key Laboratory of Horticultural Crop Biology and Genetic Improvement (East China),
Ministry of Agriculture/College of Horticulture, Nanjing Agricultural University, Nanjing 210095, P.R.China
2、Department of Plant Sciences, North Dakota State University, Fargo, ND 58105, USA

收稿日期:2013-07-26 出版日期:2014-09-22 发布日期:2014-09-24
通讯作者: LIU Li-wang, Tel: +86-25-84395563, Fax: +86-25-84395266, E-mail: nauliulw@njau.edu.cn
作者简介:PAN Yan, E-mail: panyan87@126.com
基金资助:
This work was partially supported by grants from the National Natural Science Foundation of China (31171956, 31372064) the National Key Technologies R&D Program of China (2012BAD02B01), the Key Technologies R&D Program of Jiangsu Province, China (BE2013429), the PAPD and JASTI [CX(12)2006].

Molecular Characterization and Expression Profiles of Myrosinase Gene (RsMyr2) in Radish (Raphanus sativus L.)

PAN Yan1, XU Yuan-yuan1, ZHU Xian-wen2, LIU Zhe1, GONG Yi-qin1, XU Liang1, GONG Mao-yong1, and LIU Li-wang1

1、National Key Laboratory of Crop Genetics and Germplasm Enhancement/Engineering Research Center of Horticultural Crop Germplasm
Enhancement and Utilization, Ministry of Education/Key Laboratory of Horticultural Crop Biology and Genetic Improvement (East China),
Ministry of Agriculture/College of Horticulture, Nanjing Agricultural University, Nanjing 210095, P.R.China
2、Department of Plant Sciences, North Dakota State University, Fargo, ND 58105, USA

Received:2013-07-26 Online:2014-09-22 Published:2014-09-24
Contact: LIU Li-wang, Tel: +86-25-84395563, Fax: +86-25-84395266, E-mail: nauliulw@njau.edu.cn
About author:PAN Yan, E-mail: panyan87@126.com
Supported by:
This work was partially supported by grants from the National Natural Science Foundation of China (31171956, 31372064) the National Key Technologies R&D Program of China (2012BAD02B01), the Key Technologies R&D Program of Jiangsu Province, China (BE2013429), the PAPD and JASTI [CX(12)2006].

摘要/Abstract

摘要： Myrosinase is a defense-related enzyme and is capable of hydrolyzing glucosinolates into a variety of compounds, some of which are toxic to pathogens and herbivores. Many studies revealed that a number of important vegetables or oil crops contain the myrosinase-glucosinolate system. However, the related promoter and genomic DNA sequences as well as expression profiles of myrosinase gene remain largely unexplored in radish (Raphanus sativus). In this study, the 2 798 bp genomic DNA sequence, designated as RsMyr2, was isolated and analyzed in radish. The RsMyr2 consisting of 12 exons and 11 introns reflected the common gene structure of myrosinases. Using the genomic DNA walking approach, the 5´-flanking region upstream of RsMyr2 with length of 1 711 bp was successfully isolated. PLACE and PlantCARE analyses revealed that this upstream region could be the promoter of RsMyr2, which contained several basic cis-regulatory elements including TATA-box, CAAT-box and regulatory motifs responsive to defense and stresses. Furthermore, recombinant pET-RsMyr2 protein separated by SDS-PAGE was identified as myrosinase with mass spectrometry. Real-time PCR analysis showed differential expression profiles of RsMyr2 in leaf, stem and root at different developmental stages (e.g., higher expression in leaf at cotyledon stage and lower in flesh root at mature stage). Additionally, the RsMyr2 gene exhibited up-regulated expression when treated with abscisic acid (ABA), methyl jasmonate (MeJA) and hydrogen peroxide (H2O2), whereas it was down-regulated by wounding (WO) treatment. The findings indicated that the expression of RsMyr2 gene was differentially regulated by these stress treatments. These results could provide new insight into elucidating the molecular characterization and biological function of myrosinase in radish.

关键词: radish , RsMyr2 , promoter , heterologous expression , qRT-RCR

Abstract: Myrosinase is a defense-related enzyme and is capable of hydrolyzing glucosinolates into a variety of compounds, some of which are toxic to pathogens and herbivores. Many studies revealed that a number of important vegetables or oil crops contain the myrosinase-glucosinolate system. However, the related promoter and genomic DNA sequences as well as expression profiles of myrosinase gene remain largely unexplored in radish (Raphanus sativus). In this study, the 2 798 bp genomic DNA sequence, designated as RsMyr2, was isolated and analyzed in radish. The RsMyr2 consisting of 12 exons and 11 introns reflected the common gene structure of myrosinases. Using the genomic DNA walking approach, the 5´-flanking region upstream of RsMyr2 with length of 1 711 bp was successfully isolated. PLACE and PlantCARE analyses revealed that this upstream region could be the promoter of RsMyr2, which contained several basic cis-regulatory elements including TATA-box, CAAT-box and regulatory motifs responsive to defense and stresses. Furthermore, recombinant pET-RsMyr2 protein separated by SDS-PAGE was identified as myrosinase with mass spectrometry. Real-time PCR analysis showed differential expression profiles of RsMyr2 in leaf, stem and root at different developmental stages (e.g., higher expression in leaf at cotyledon stage and lower in flesh root at mature stage). Additionally, the RsMyr2 gene exhibited up-regulated expression when treated with abscisic acid (ABA), methyl jasmonate (MeJA) and hydrogen peroxide (H2O2), whereas it was down-regulated by wounding (WO) treatment. The findings indicated that the expression of RsMyr2 gene was differentially regulated by these stress treatments. These results could provide new insight into elucidating the molecular characterization and biological function of myrosinase in radish.

Key words: radish , RsMyr2 , promoter , heterologous expression , qRT-RCR

PAN Yan1, XU Yuan-yuan1, ZHU Xian-wen2, LIU Zhe1, GONG Yi-qin1, XU Liang1, GONG Mao-yong1, and LIU Li-wang1. Molecular Characterization and Expression Profiles of Myrosinase Gene (RsMyr2) in Radish (Raphanus sativus L.)[J]. Journal of Integrative Agriculture, 2014, 13(9): 1877-1888.

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Molecular Characterization and Expression Profiles of Myrosinase Gene (RsMyr2) in Radish (Raphanus sativus L.)

Molecular Characterization and Expression Profiles of Myrosinase Gene (RsMyr2) in Radish (Raphanus sativus L.)

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